The members of the HER family of receptor tyrosine kinases are important mediators of cell growth, differentiation, migration and survival. The receptor family includes four distinct members including epidermal growth factor receptor (EGFR, ErbB1, or HER1), HER2 (ErbB2 or p185<neu>), HER3 (ErbB3) and HER4 (ErbB4). The members of the EGFR family are closely related single-chain modular glycoproteins with an extracellular ligand binding region, a single transmembrane domain and an intracellular tyrosine kinase, followed by specific phosphorylation sites which are important for the docking of downstream signaling proteins.
The extracellular regions of the HER receptor family contain two homologous ligand binding domains (domains 1 and 3) and two cysteine-rich domains (domains 2 and 4), which are important for receptor dimerization. In the absence of a ligand, HER receptors normally exist as inactive monomers, known as the “tethered” structure, which is characterized by close interaction of domain 2 and 4. Ligand binding to the extracellular domain initiates a conformational rearrangement, exposing the dimerization domains 2 and 4. Therefore, binding of growth factors to HER receptors induces conformational changes that allow receptor dimerization. After extracellular receptor dimerization, transmembrane helices switch to an active conformation that enables the intracellular kinase domains to trans-auto-phosphorylate each other. This phosphorylation event allows the recruitment of specific downstream signaling proteins.
Epidermal Growth factor receptor 1, (EGFR), has been causally implicated in human malignancy. In particular, increased expression of EGFR has been observed in breast, bladder, lung, head, neck and stomach cancer as well as glioblastomas.
Human epidermal growth factor receptor 2 (HER2, also known as ErbB2 or Neu; UniProtKB/Swiss-Prot No. P04626) consists of 1233 amino acids and is structurally similar to EGFR, with an extracellular domain consisting of four subdomains 1-4, a transmembrane domain, a juxtamembrane domain, an intracellular cytoplasmic tyrosine kinase and a regulatory C-terminal region. The structure of HER2's extracellular region is different in important aspects from the other EGF receptors, however. In the other EGF receptors, in a non-activated state, domain 2 binds to domain 4. Upon binding to domains 1 and 3, the activating growth factor (ligand) selects and stabilizes a conformation that allows a dimerization arm to extend from domain 2 to interact with an ErbB dimer partner. HER2, on the other hand, has a fixed conformation that resembles the ligand-activated state of the other receptor members: the domain 2-4 interaction is absent and the dimerization loop in domain 2 is continuously exposed. HER2 is activated via formation of heterodimeric complexes with other ErbB family members and thereby indirectly regulated by EGFR and HER3 ligands. HER2 is the preferred heterodimerization partner of the three other ErbB receptors, enhancing the affinity of the other ErbB receptors for their ligands by slowing down the rate of ligand-receptor complex dissociation, whereby HER2 enhances and prolongs signaling.
An excess of HER2 on the cell surface causes transformation of epithelial cells from multiple tissues. Amplification of the human homolog of the neu gene (also known as HER2) is observed in breast and ovarian cancers and correlates with a poor prognosis (U.S. Pat. No. 4,968,603). Overexpression of HER2 has also been observed in other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder.
Antibodies Targeting HER2
Drebin and colleagues have raised antibodies against the rat neu gene product, p185<neu>disclosed in U.S. Pat. No. 6,733,752 (B1).
Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989) describe the generation of a panel of HER2 antibodies which were characterized using the human breast tumor cell line SkBr-3. Using a cell proliferation assay, maximum inhibition was obtained with an antibody called 4D5. The antibody 4D5 was further found to sensitize HER2-overexpressing breast tumor cell lines to the cytotoxic effects of TNF-[alpha]; see also U.S. Pat. No. 5,677,171. A recombinant humanized version of the murine HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, trastuzumab or HERCEPTIN; U.S. Pat. No. 5,821,337) is clinically active in patients with HER2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy. Herceptin is approved in combination with chemotherapy for use in patients with HER2-positive metastatic stomach (gastric) cancer.
Herceptin is widely used for the treatment of patients with early as well as metastatic breast cancer whose tumors overexpress HER2 protein and/or have HER2 gene amplification. The treatment of breast cancer patients with Herceptin/trastuzumab is, for example, recommended and now routine for patients having HER2-positive disease; see US 2002/0064785, US 2003/0170234A1, US2003/0134344 and US 2003/0147884. The prior art thus focuses on the eligibility of breast cancer patients for trastuzumab/Herceptin therapy based on a high HER2 protein expression level (e.g. defined as HER2(3+) by immunohistochemistry (IHC)). HER2-positive disease in breast cancer is defined to be present if a high HER2 (protein) expression level is detected by immunohistochemical methods (e.g. HER2 (+++) or as HER2 gene amplification (e.g. a HER2 gene copy number higher than 4 copies of the HER2 gene per tumor cell) or both, found in samples obtained from the patients such as breast tissue biopsies or breast tissue resections or in tissue derived from metastatic sites. One frequently applied method for detecting HER2 overexpression and amplification at the gene level is fluorescence in situ hybridization (FISH), which is also described in US2003/0152987, Cohen et al.
Pertuzumab, a humanized antibody, is the first of a new class of agents known as HER dimerization inhibitors (HD's). Pertuzumab binds to HER2 at its dimerization domain, thereby inhibiting its ability to form active heterodimer receptor complexes, thus blocking the downstream signal cascade that ultimately results in cell growth and division. Pertuzumab is directed against the extracellular domain 2 of HER2. In contrast to trastuzumab, which acts by binding to domain 4 of HER2, pertuzumab is a HER dimerization inhibitor which inhibits dimerization of HER2 with HER3 and the other members of the EGFR receptor family in the presence of the respective activating ligands. By blocking complex formation, pertuzumab prevents the growth-stimulatory effects and cell survival signals activated by ligands of HER1, HER3 and HER4. Pertuzumab has been approved by the FDA under the name Perjeta for treatment in combination with trastuzumab and docetaxel for patients with HER2-positive metastatic breast cancer, who have not received prior anti-HER2 therapy or chemotherapy for metastatic disease. Pertuzumab is a fully humanized recombinant monoclonal antibody based on the human IgG1([kappa]) framework sequences. Patent publications concerning pertuzumab and selection of patients for therapy therewith include: US20060073143 (A1); US2003/0086924; US2004/0013667A1, and US2004/0106161.
For trastuzumab, while known to show clinical benefits in terms of e.g. prolonged survival in combination with chemotherapy compared to chemotherapy alone, a majority of HER2 positive breast cancer patients were nevertheless found to be non-responders (45% overall response rate for Herceptin+chemotherapy vs. 29% for chemotherapy alone).
Thus, while monoclonal antibody therapy directed against HER2 has been shown to provide improved treatment in e.g. metastatic breast cancers that overexpress HER2, there is still considerable room for improvement.
Non-Antibody Scaffolds Targeting HER2
Alternative targeting proteins have been proposed recently, which are more diverse in molecular structure than human immunoglobulin-derived antibody fragments and antibody-derived constructs and formats, and thus allow additional molecular formats by creating heterodimeric and multimeric assemblies, leading to new biological functions. A number of such targeting proteins have been described (reviewed in (Binz et al., Nat. Biotech 2005, Vol 23:1257-1268)). Non-limiting examples of such targeting proteins are camelid antibodies, protein scaffolds derived from protein A domains (termed “Affibodies”, Affibody AB), tendamistat (an alpha-amylase inhibitor, a 74 amino acid beta-sheet protein from Streptomyces tendae), fibronectin, lipocalin (“Anticalins”, Pieris), T-cell receptors, ankyrins (designed ankyrin repeat proteins termed “DARPins”, Univ. Zurich and Molecular Partners; see US20120142611 (A1)), A-domains of several receptors (“Avimers”, Avidia) and PDZ domains, fibronectin domains (FN3) (“Adnectins”, Adnexus), consensus fibronectin domains (“Centyrins”, Centyrex/Johnson&Johnson) and Ubiquitin (“Affilins”, SCIL Proteins) and knottins (Moore and Cochrane, Methods in Enzymology 503 (2012), 223-251 and references cited therein).
From these proteins, multimeric and multispecific assemblies can be constructed (Caravella and Lugovskoy, Current Opinions in Chemical Biology 2010, 14:520-528; Vanlandschoot et al. Antiviral Research 2011 92:389-407; Lofblom et al. 2011 Current Opinion in Biotechnology 2011 22:843-848, Boersma et al. 2011 Curr. Opin. Biotechnol. 22:849-857). It is also possible to fuse these and other peptidic domains to antibodies to create so-called Zybodies (Zyngenia Inc., Gaithersburg, Md.).
All of these scaffolds, with different inherent properties, have in common that they can be directed to bind specific epitopes, by using selection technologies well known to practitioners in the field (Binz et al., Nat. Biotech 2005, 23:1257-1268).
For example, the different individual domains of HER2 can be individually expressed in insect cells, using a baculovirus expression system, as demonstrated for domain 1 and domain 4 (Frei et al., Nat Biotechnol. 2012 30:997-1001). Thereby, it is guaranteed that binders selected will be directed towards the domain of interest. The HER2 domains can then be biotinylated as previously described (Zahnd et al., (2006). Selection and characterization of HER2 binding-designed ankyrin repeat proteins. J. Biol. Chem. 281), and thus be immobilized on streptavidin-coated magnetic beads or on microtiter plates coated with streptavidin or neutravidin (Steiner et al. (2008) J. Mol. Biol. 382, 1211-1227); (Zahnd et al. (2007) J. Mol. Biol. 369, 1015-1028.)). The HER2 domains so immobilized can then serve as targets for diverse protein libraries in either phage display or ribosome display format. A large variety of different antibody libraries has been published (Mondon P. et al., Human antibody libraries: a race to engineer and explore a larger diversity. Frontiers in Bioscience. 13:1117-1129, 2008.) and the technology of selecting binding antibodies is well known to the practitioners of the field. Phage display is a suitable format for antibody fragments (Fab fragments, scFv fragments or single domain antibodies s) (Hoogenboom H R. Nature Biotechnology. 23(9):1105-1116, 2005 September) and any other scaffold that contain disulfide bonds, but it can also be used for scaffolds not containing disulfide bonds (e.g., Steiner et al. (2008) J. Mol. Biol. 382, 1211-1227)(Rentero et al. Chimia. 65(11):843-5, 2011., Skerra A. Current Opinion in Biotechnology. 18(4):295-304, 2007 August). Similarly, ribosome display can be used for antibody fragments (Hanes et al. (2000), Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat. Biotechnol. 18, 1287-1292) and for other scaffolds (Zahnd et al. (2007). Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat. Methods 4, 269-279; Zahnd et al. (2007) J. Mol. Biol. 369, 1015-28.). A third powerful technology is yeast display (Pepper et al., Combinatorial Chemistry & High Throughput Screening. 11(2):127-134, 2008 February). In this case a library of the binding protein of interest is displayed on the surface of yeast, and the respective domain of HER2 is either directly labeled with a fluorescent dye or its his tag is detected with an anti-histag antibody, which is in turn detected with a secondary antibody. Such methods are well known to the practitioners in the field (Boder et al., Yeast surface display for directed evolution of protein expression, affinity, and stability, Methods in Enzymology. 328:430-44, 2000.).
Another possibility of engineering represents the connection of those binders to create bispecific or higher multivalent binding molecules. Such connection can be achieved genetically by fusions of two or more of these binding molecules or chemically by crosslinking separately expressed molecules, or by adding a dimerization domain include separate dependent claims for each or any combination thereof (see, e.g. Stefan et al. (2011) J. Mol. Biol. 413:826-843; Boersma et al. (2011) J. Biol. Chem. 286: 41273-41285)).
A bispecific anti-HER2 camelidae antibody construct (Bispecific Nanobody) is shown in US20110059090 (A1). The document relates to a bispecific molecule that simultaneously targets HER2 at the extracellular domain 2, defined by competition with pertuzumab, and domain 4, defined by competition with trastuzumab. This molecule has been described to exhibit stronger anti-proliferative activity than trastuzumab (Herceptin) in a direct comparison in an in vitro cell culture model using the cell line SkBr3.
Due to the absence of any known HER2-specific ligand, current HER2 targeting strategies aim to block the dimerization of the receptor by binding to the interaction interface. Today's knowledge of HER2 receptor dimerization is mostly based on the crystal structure of the ligand-bound form of the EGFR homodimer, which is broadly accepted as the active mode of all EGF receptor family members (Garret et al. (2002) Cell 110, 763-773). The two EGFR molecules show a back-to-back interaction. Extending these findings to HER2 and its possible interaction with other members of the EGFR family, one interaction interface is present on domain 2 of the extracellular part of HER2. Pertuzumab binds to domain 2 and is indeed known to block receptor interaction at this interface. Another known interaction is present on domain 4 of the extracellular part of HER2. This interaction interface is presumably blocked by trastuzumab. Yet both antibodies, trastuzumab and pertuzumab, even when simultaneously applied, are not able to block all HER2 interactions to completeness. The interaction of the extracellular part and the kinase domain of HER2 are thought to be linked in such a way as to allow some residual interactions even in the trastuzumab- and pertuzumab-blocked state, which is in accordance with crystal structure data (Lu et al. (2010) Mol. Cell. Biol. (22):5432-5443). The bispecific ligand mentioned above that binds both epitopes (pertuzumab and trastuzumab) simultaneously (US20110059090 A1) reduces the cell growth in a cell culture model by approx. 50%, in comparison to a reduction of about 40% effected by trastuzumab. This same effect, however, can also be achieved by treating with the mixture of trastuzumab and pertuzumab.
In view of the above mentioned state of the art, the objective of the present invention is to provide improved means and methods for targeting the HER2 protein for use in therapy of cancer. This objective is attained by the subject-matter of the independent claims.